This investigator's laboratory is specifically concerned with the structural and functional characterization of eukaryotic translation initiation factors required for the in vitro formation of a functional 80S ribosomal initiation complex, and the interaction of these protein factors with ribosomes, initiator Met-tRNA, GTP and mRNA during initiation complex formation. In the current grant proposal the following areas will be pursued. (a) The mechanism of elF-5-mediated hydrolysis of GTP bound to the 40S initiation complex will be characterized. elF-5 catalyzes the following reaction [40S-AUG-Met-tRNA1,.elF-2-GTP + 60Self-5 [80SAUGMET-tRNA]+[elF- 2GDP]+P. The mammalian cDNA and the yeast gene encoding elF-5 have been cloned and sequenced in the PI's laboratory. Analysis of the derived amino acid sequence reveals the presence of sequence motifs characteristics of proteins of the GTPase superfamily. The successful expression of functionally active recombinant rat elF-5 in Escherichia coli will allow the PI's laboratory to investigate the function of these motifs in elF-5. Site-directed mutagenesis of these sequences will be carried out to examine their role in the hydrolysis of GTP bound to elF-2 on the 40S initiation complex and to study the significance of the specific association of elF-5 with elF-2 in the hydrolysis reaction. (b) elF-5 is phosphorylated at one or two serine residues in vivo. The sequence of the tryptic phosphopeptides will be determined. Using purified bacterially-expressed recombinant elF-5 as a substrate in vitro, the protein kinase responsible for phosphorylating elF-5 in vivo will be purified and characterized. The effect of phosphorylation of elF-5 on its biological activity will be investigated. (c) Using an assay that measures elF-3-dependent transfer of Met-tRNA, elF-2-GTP to 40s subunits to form the 40s preinitiation complex, (40S elF-3lF-3Met-tRNA, elF-2-GTP), the multisubunit elF-3 protein (120,67, 43,42,36 kDa) has been purified and antibodies raised against each of the subunits. Evidence indicates that only the 120 kDa subunit is required for the transfer of Met-tRNA, elf-2-GTP to 40S subunits in the absence of mRNA. The mammalian cDNA for the 120 kDa protein will be cloned and expressed and the function of this protein in the 40S transfer reaction will be characterized. The role of the others subunits of elF-3 in the binding of mRNA to the 40S preinitiation complex will be studied. (d) The interaction of elF-5 with the 40S initiation complex formed with mRNA and the requirements for the subsequent joining of 60S ribosomal subunits to the 40S initiation complex will be investigated. Using the two hybrid system in yeast cells, a mammalian cDNA expression library will be screened to identify proteins that interact wit elF-5. The function of these putative proteins in the initiation reaction will be studied.